Field of the Invention
The present invention relates to a method for obtaining a natural variant of an enzyme and relates to super thermostable cellobiohydrolase obtained by the method.
Priority is claimed on Japanese Patent Application No. 2014-050083, filed on Mar. 13, 2014, the content of which is incorporated herein by reference.
Description of Related Art
Lignocellulose biomass is abundantly available on the earth, and for this reason, biofuel such as ethanol of which the raw material is lignocellulose is expected to become transportation energy replacing fossil fuels. For converting lignocellulose into ethanol, hydrolysis of cellulose or hemicelluloses needs to be performed. The cellulose hydrolysis is categorized into two methods including sulfuric acid hydrolysis and enzymatic hydrolysis. The enzymatic hydrolysis using glycoside hydrolases, which are collectively called cellulase enzymes in general, has advantages in that it does not generate sulfuric acid sludge unlike the sulfuric acid hydrolysis, consumes a small amount of energy, and results in high yield without causing excessive degradation of lignocellulose.
The cellulose constituting plant cell walls has a crystal structure in which the cellulose molecules are combined with each other through a strong hydrogen bond, and accordingly, the cellulose is practically not dissolved in water. Meanwhile, the enzymatic hydrolysis of cellulose is a solid-liquid reaction. During the solid-liquid reaction, the hydrolysis of the crystalline cellulose occurs on the solid surface, and consequentially, hydrolysis efficiency resulting from the enzyme is markedly low. For instance, cellobiohydrolase (CBH) as a main cellulose hydrolase results in a hydrolysis rate that is lower than that of other polysaccharide lyases by a single digit or double digits. Accordingly, the enzyme needs to be used in a large amount for hydrolysis of cellulose biomass, and this is a main factor causing a rise in the cost of cellulose-based biofuels. In order to reduce the cost of cellulose ethanol, the hydrolysis efficiency of the cellulase enzyme needs to be greatly improved.
As one of the methods for improving the enzyme efficiency, thermostability of the enzyme protein is improved. In order to improve the thermostability of the enzyme protein, various means for reengineering the enzyme by introduction of amino acid mutation, such as a directed evolution (DE) method in which amino acid sequences are randomly substituted, a site-directed mutagenesis (SDM) method in which amino acid substitution is performed only in a specific site, and a method in which a chimeric sequence is formed by cleaving a plurality of genes at several sites and then shuffling the fragments, have been tried.
Theoretically, the enzyme modification performed by the DE method has poor efficiency. This is because a so-called problem of“combinatorial explosion” occurs in which the number of variants increases exponentially as the number of the cases of point mutation increases. In order to obtain optimal amino acid sequences, it is necessary to construct mutant libraries for combinations of all amino acid residues and to perform a functional amino assay, however, this is impossible in principle. Furthermore, while most of the mutations are loss-of-function type mutation resulting in loss of function, neutral mutation or gain-of-function type mutation by which advantageous functions are obtained is extremely rare, and also hinders the DE method from efficiently searching for optimal amino acid sequences.
Because an enormous number of mutant libraries should be screened to obtain effective mutants, and there is no effective high-throughput screening method or a stable gene expression system (for example, see Himmel et al., Current Opinion in Biotechnology, 1999, vol. 10, p. 358˜364.), the function of the cellulase enzyme has not been sufficiently improved by the DE method.
For example, Nakazawa et al. reported that they reengineered endoglucanase EGIII of a wood-rotting fungus, Trichoderma reesei, by the DE method (see Nakazawa et al., Applied Microbiology and Biotechnology, 2009, vol. 83, p. 649˜657). The EGIII is a protein which is constituted with 218 amino acid residues and has a relatively small molecular weight. However, even in the case of such a small size protein, the number of variants obtained by introducing mutation causing amino acid substitution into any single site becomes 4,360 (20×218); the number of variants obtained by introducing mutation into any two sites becomes 9,460,000 (20×20×218×217÷2); and the number of variants obtained by introducing mutation into any three sites becomes 13,624,130,000 (20×20×20×(218×217×216÷6)) due to the combinatorial explosion.
In order to obtain variants in which mutation has occurred in only two to three sites, tens of millions to tens of billions of mutant libraries need to be constructed, and functional screening thereof needs to be performed. Therefore, this method is not practical. Although Nakazawa et al. performed functional screening for a total of 11,000 first- and second-generation mutants, the optimal temperature thereof was increased by only +5° C., and they did not achieve great improvement in thermostability. Naturally, if the scale of the mutant library is small, effective mutants are not discovered. Moreover, from 9,000 colonies of the mutant libraries of the first generation having undergone the functional screening, the variants having CMC hydrolytic activity are obtained in only 46 colonies. The remaining 8,954 mutants are considered to have lost their function due to amino acid mutation or have gene expression failure, and most of the random mutations caused by erroneous PCR of the DE method result in the loss of function of the enzyme protein. In this way, because the DE method produces a large amount of loss-of-function type mutants, the screening efficiency thereof is extremely poor.
The gene shuffling method has not succeeded in obtaining chimeric genes having thermostability higher than that of parental genes thereof. For example, Heinzelman et al. proposed a method for improving the function of an enzyme by creating chimeric genes by means of cleaving a plurality of cellobiohydrolase genes at several sites and shuffling the fragments thereof (see Heinzelman et al., Proceedings of the National Academy of Sciences of the United States of America, 2009, vol. 106, p. 5610˜5615). According to this method, in order to improve the thermostability of an exoglucanase CBH II of Trichoderma reesei, genes of the enzyme CBH II of thermophilic filamentous fungi Humicola insolens and Chaetomium thermophilum are combined together by gene shuffling, thereby preparing a chimeric enzyme. However, the thermostability of the chimeric enzyme is not higher than that of CBH II of the thermophilic filamentous fungus Chaetomium thermophilum as a parent thereof and the gene shuffling is ineffective.
While the DE method is a method for causing amino acid substitution by random mutation, the SDM method is a method for causing mutation such as substitution, deletion, or insertion of amino acids based on the three-dimensional structure data of enzyme proteins. Unlike the DE method, the SDM method does not require screening of an enormous number of mutants. However, generally, it is difficult to identify the site involved in the improvement of the function of an enzyme, and it is rare for the SDM method to exert a marked effect on the improvement of the cellulase enzyme.
For example, in cellobiohydrolase, the N terminal and C terminal thereof have a loop structure and form a tunnel structure covering the active center. Based on such a structure, the SDM method for substituting the amino acid inside the loop has been tried. In order to improve the thermostability, Zhang et al. estimated the three-dimensional structure of a cellobiohydrolase TfCel6B of a thermophilic actinomycete Thermobifida fusca, and introduced a disulfide bond into the loop of the N terminal and the C terminal forming the tunnel structure of the active site by double mutation. However, contrary to their expectation, in the obtained variant, a temperature T50 at which the enzymatic activity halves was reduced by 10° C. (see Zhang et al., European Journal of Biochemistry, 2000, vol. 267, p. 3101˜3115). Zhang at al. reported that although they substituted glycine residues at 4 sites with alanine, serine, and proline, no cellubiohydrolase of which the thermostability was improved by mutation was obtained, and the thermostability was reduced by 5° C. to 10° C. in most of the mutants.
Furthermore, Wohlfahrt et al. reported that in order to improve heat stability of a cellubiohydrolase TrCel6A belonging to a GH6 family of the filamentous fungus T. reesei, they manufactured a variant by introducing mutation into amino acid residues positioned on the N terminal loop and the C terminal loop and amino acids residues positioned in the near of the loops, and as a result, a thermal denaturation temperature (Tm) of the variant increased at a pH of 7.0, but the Tm value decreased at a pH of 5.0 which is the optimal pH of wild type TrCel6A (see Wohlfahrt et al., Biochemistry, 2003, vol. 42, p. 10095˜10103). Meanwhile, in order to stabilize an exo-loop structure forming a tunnel loop of the active center of a cellubiohydrolase TrCel7A belonging to a GH7 family of the filamentous fungus T. reesei, von Ossowski et al. prepared a variant obtained by introducing an SS (disulfide) bond into the loop, but in this variant, the improvement of the thermostability was not observed at all (see von Ossowski et al., Journal of Molecular Biology, 2003, vol. 333, p. 817˜829). As described above, the attempt at introducing a hydrogen bond or an SS bond into the C terminal loop forming the loop structure, which is considered to be deeply involved with the function of the cellobiohydrolase enzyme, has been made in various ways, but up to now, the attempt has not yet led to success in improvement of the function of the enzyme, such as the improvement of thermostability.
From the comparison between homologous genes of species, it has been found that there is a domain which is well preserved and a domain into which amino acid mutation is frequently introduced. The preserved domain is called a “consensus sequence” because it is a common sequence that characterizes a protein of a specific category. If amino acid residues in the consensus sequence are mutated, the enzyme easily loses its function. It is considered that for this reason, the site has been well preserved through a long evolutionary process. Accordingly, the mutation that causes amino acid mutation in the consensus sequence is highly likely to result in loss of function. Therefore, the SDM method has been tried which causes mutation in a protein except for the consensus sequence. However, this method can only be applied to enzymes of which the three-dimensional structure of protein is determined by X-ray crystal structure analysis, and there are only a few enzyme proteins of which the three dimensional structure has been identified.